scholarly journals Comparative Analyses of Single-Cell Transcriptomic Profiles between In Vitro Totipotent Blastomere-like Cells and In Vivo Early Mouse Embryonic Cells

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3111
Author(s):  
Po-Yu Lin ◽  
Denny Yang ◽  
Chi-Hsuan Chuang ◽  
Hsuan Lin ◽  
Wei-Ju Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Embryonic Cells ◽  
Cell Stage ◽  
Developmental Potential ◽  
Functional Features ◽  
Early Mouse ◽  
Extraembryonic Tissues

The developmental potential within pluripotent cells in the canonical model is restricted to embryonic tissues, whereas totipotent cells can differentiate into both embryonic and extraembryonic tissues. Currently, the ability to culture in vitro totipotent cells possessing molecular and functional features like those of an early embryo in vivo has been a challenge. Recently, it was reported that treatment with a single spliceosome inhibitor, pladienolide B (plaB), can successfully reprogram mouse pluripotent stem cells into totipotent blastomere-like cells (TBLCs) in vitro. The TBLCs exhibited totipotency transcriptionally and acquired expanded developmental potential with the ability to yield various embryonic and extraembryonic tissues that may be employed as novel mouse developmental cell models. However, it is disputed whether TBLCs are ‘true’ totipotent stem cells equivalent to in vivo two-cell stage embryos. To address this question, single-cell RNA sequencing was applied to TBLCs and cells from early mouse embryonic developmental stages and the data were integrated using canonical correlation analyses. Differential expression analyses were performed between TBLCs and multi-embryonic cell stages to identify differentially expressed genes. Remarkably, a subpopulation within the TBLCs population expressed a high level of the totipotent-related genes Zscan4s and displayed transcriptomic features similar to mouse two-cell stage embryonic cells. This study underscores the subtle differences between in vitro derived TBLCs and in vivo mouse early developmental cell stages at the single-cell transcriptomic level. Our study has identified a new experimental model for stem cell biology, namely ‘cluster 3’, as a subpopulation of TBLCs that can be molecularly defined as near totipotent cells.

2 THE IN VIVO DEVELOPMENTAL POTENTIAL OF PORCINE SKIN-DERIVED PROGENITORS

2012 ◽  
Vol 24 (1) ◽  
pp. 112 ◽  
Author(s):  
M. T. Zhao ◽  
X. Yang ◽  
K. Lee ◽  
J. Mao ◽  
J. M. Teson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lineage ◽  
Pcr Amplification ◽  
Cell Types ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Germ Layers ◽  
Developmental Potential

Skin-derived progenitors (SKP) are capable of generating both neural and mesodermal progeny in vitro: neurons, Schwann cells, adipocytes, osteocytes and chondrocytes, thus exhibiting characteristics similar to embryonic neural crest stem cells. SKP show distinct transcriptional profiles when compared with neurospheres/neural stem cells in the central nervous system (CNS) and skin-derived fibroblasts, indicating a novel type of multipotent stem cell derived from the dermis of the skin. However, it remains unclear whether SKP cells can produce ectoderm and mesoderm lineages or other germ layers in vivo, although oocyte-like structures can be induced from porcine SKP in vitro. Embryonic chimeras are a well-established tool for investigating cell lineage determination and cell potency through normal embryonic development. Thus the purpose of this study was to investigate the in vivo developmental potential of porcine SKP by chimera production. Porcine SKP cells and fibroblasts were isolated from the back skin of Day 35 to 50 GFP transgenic fetuses. Individual cells or clusters of male GFP transgenic SKP and skin-derived GFP-expressing fibroblasts were injected into pre-compact in vitro-fertilized (IVF) embryos, respectively and then transferred into corresponding surrogates 24 h post-injection. Additional injected embryos were cultured in PZM3 medium for another 2 days until the blastocyst stage and subsequently stained with Hoechst 33342. Interestingly, in some of the chimeras the injected SKP cells migrated and dispersed into different locations of the host blastocysts, whereas in others they remained as a cluster of cells within the chimeric blastocysts. In contrast, the fibroblast cells were not observed to spread around the host blastocysts. Two chimeric fetuses were recovered at the middle of gestation and a litter of viable piglets was born. Genomic DNA was extracted from various tissues of chimeric piglets and subjected to PCR amplification. Two chimeric fetuses and 2 out of 6 piglets carried the GFP transgene in SKP-derived chimeras, but GFP was not present in the fibroblast-derived chimeric fetuses (n = 6). Surprisingly, the GFP transgene was present in various tissues of two SKP-derived chimeric piglets, including lung, heart, liver, artery, kidney, brain, skin, muscle, gut, ovary, pancreas and stomach, thus representing the 3 germ layers (ectoderm, mesoderm and endoderm). In addition, SRY was detected in several tissues of the two GFP-positive female chimeric piglets, confirming the chimerism of these piglets. Therefore, it appears that porcine SKP can contribute to various cell types of the 3 germ layers and have a broader developmental potency than previously expected. Alternatively, pre-compact (4-cell and 8-cell stage) embryos may provide a unique environment for reprogramming skin-derived progenitors into a more primitive state by the process of embryonic compaction. This study was funded by NIH National Center for Research Resources (R01RR013438) and Food for the 21st Century at the University of Missouri.

scholarly journals Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors

2019 ◽  
Author(s):  
Rongqun Guo ◽  
Fangxiao Hu ◽  
Qitong Weng ◽  
Cui Lv ◽  
Hongling Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Time Window ◽  
Immune Surveillance ◽  
Cell Stage ◽  
Immunodeficient Mice ◽  
Cell Transcriptome ◽  
T Lymphopoiesis

ABSTRACTAchievement of immunocompetent and therapeutic T lymphopoiesis from pluripotent stem cells is a central aim in T cell regenerative medicine. To date, preferentially regenerating T lymphopoiesis in vivo from pluripotent stem cells (PSC) remains a practical challenge. Here we documented that synergistic and transient expression of Runx1 and Hoxa9 restricted in the time window of endothelial to hematopoietic transition and hematopoietic maturation stages induced in vitro from PSC (iR9-PSC) preferentially generated engraftable hematopoietic progenitors capable of homing to thymus and developing into mature T (iT) cells in primary and secondary immunodeficient recipients. Single-cell transcriptome and functional analyses illustrated the cellular trajectory of T lineage induction from PSC, unveiling the T-lineage specification determined at as early as hemogenic endothelial cell stage and identifying the bona fide pre-thymic progenitors. The iT cells distributed normally in central and peripheral lymphoid organs and exhibited abundant TCRαβ repertoire. The regenerative T lymphopoiesis rescued the immune-surveillance ability in immunodeficient mice. Furthermore, gene-edited iR9-PSC produced tumor-specific-T cells in vivo that effectively eradicated tumor cells. This study provides insight into universal generation of functional and therapeutic T lymphopoiesis from the unlimited and editable PSC source.

The effect of dual inhibition of Ras–MEK–ERK and GSK3β pathways on development of in vitro cultured rabbit embryos

Zygote ◽  
2020 ◽  
Vol 28 (3) ◽  
pp. 183-190 ◽  
Author(s):  
Babett Bontovics ◽  
Pouneh Maraghechi ◽  
Bence Lázár ◽  
Mahek Anand ◽  
Kinga Németh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Activin A ◽  
Mouse Strains ◽  
Suitable Model ◽  
Embryonic Cells ◽  
Cell Stage ◽  
Human Ipscs ◽  
Dual Inhibition ◽  
Rabbit Embryos

SummaryDual inhibition (2i) of Ras–MEK–ERK and GSK3β pathways enables the derivation of embryo stem cells (ESCs) from refractory mouse strains and, for permissive strains, allows ESC derivation with no external protein factor stimuli involvement. In addition, blocking of ERK signalling in 8-cell-stage mouse embryos leads to ablation of GATA4/6 expression in hypoblasts, suggesting fibroblast growth factor (FGF) dependence of hypoblast formation in the mouse. In human, bovine or porcine embryos, the hypoblast remains unaffected or displays slight-to-moderate reduction in cell number. In this study, we demonstrated that segregation of the hypoblast and the epiblast in rabbit embryos is FGF independent and 2i treatment elicits only a limited reinforcement in favour of OCT4-positive epiblast populations against the GATA4-/6-positive hypoblast population. It has been previously shown that TGFβ/Activin A inhibition overcomes the pervasive differentiation and inhomogeneity of rat iPSCs, rat ESCs and human iPSCs while prompting them to acquire naïve properties. However, TGFβ/Activin A inhibition, alone or together with Rho-associated, coiled-coil containing protein kinase (ROCK) inhibition, was not compatible with the viability of rabbit embryos according to the ultrastructural analysis of preimplantation rabbit embryos by electron microscopy. In rabbit models ovulation upon mating allows the precise timing of progression of the pregnancy. It produces several embryos of the desired stage in one pregnancy and a relatively short gestation period, making the rabbit embryo a suitable model to discover the cellular functions and mechanisms of maintenance of pluripotency in embryonic cells and the embryo-derived stem cells of other mammals.

scholarly journals Full-term development of enucleated mouse oocytes fused with embryonic stem cells from different cell lines

Reproduction ◽  
2001 ◽  
pp. 729-733 ◽  
Author(s):  
T Amano ◽  
Y Kato ◽  
Y Tsunoda
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Formation Rate ◽  
Embryonic Stem ◽  
Clear Correlation ◽  
Developmental Potential ◽  
Mouse Oocytes

The developmental potential of enucleated mouse oocytes receiving embryonic stem cells from ten lines with either the same or different genetic backgrounds using the cell fusion method was examined in vitro and in vivo. The development of nuclear-transferred oocytes into blastocysts was high (34-88%). However, there was no clear correlation between development into blastocysts after nuclear transfer and the chimaera formation rate of embryonic stem cells. The development into live young was low (1-3%) in all cell lines and 14 of 19 young died shortly after birth. Most of the live young had morphological abnormalities. Of the five remaining mice, two died at days 23 and 30 after birth, but the other three mice are still active at days 359 (mouse 1) and 338 (mice 4 and 5) after birth, with normal fertility. However, the reasons for the abnormalities and postnatal death of embryonic stem cell-derived mice are unknown.

scholarly journals Oct4 regulates embryonic pluripotency via metabolic mechanisms and Stat3 signalling

2020 ◽  
Author(s):  
Giuliano Giuseppe Stirparo ◽  
Agata Kurowski ◽  
Stanley Eugene Strawbridge ◽  
Hannah Stuart ◽  
Thorsten Edwin Boroviak ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Specific Gene ◽  
List Type ◽  
Early Mouse

AbstractOCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 protects the pluripotent lineage from differentiation into trophoblast, we used single cell transcriptomics and quantitative immunofluorescence on blastocysts and established differentially expressed genes and pathways between control and OCT4 null cells. Activation of most pluripotency-associated transcription factors in the early mouse inner cell mass appears independent of OCT4, whereas JAK/STAT signalling requires OCT4, via activation of IL6ST. Single cell deconvolution, diffusion component and trajectory inference dissected the process of differentiation of OCT4 null cells by activating specific gene-network and transcription factors. Downregulation of glycolytic and oxidative metabolism was observed. CHIPseq analysis suggests OCT4 directly targets rate-limiting glycolytic enzymes. Concomitant with significant disruption of the STAT3 pathway, oxidative respiration is significantly diminished in OCT4 null cells. Upregulation of the lysosomal pathway detected in OCT4 null embryos is likely attributable to aberrant metabolism.Highlights and noveltyMajor pluripotency-associated transcription factors are activated in OCT4-deficient early mouse ICM cells, coincident with ectopic expression of trophectoderm markersJAK/STAT signalling is defective in OCT4 null embryosOCT4 promotes expression of KATS enzymes by means of glycolytic production of Acetyl CoA to secure chromatin accessibility for acquisition of epiblast identityOCT4 regulates the metabolic and biophysical processes required for establishment of embryonic pluripotency

scholarly journals Paracrine Signalling From SOX2-Expressing Pituitary Embryonic Cells Is Required for Terminal Differentiation of Hormone-Producing Cells

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A547-A548
Author(s):  
Saba Manshaei ◽  
Thea Willis ◽  
Dominic Withers ◽  
Jesus Gil ◽  
Cynthia Lilian Andoniadou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Rna Binding ◽  
Terminal Differentiation ◽  
Published Data ◽  
Embryonic Cells ◽  
Human Pituitary ◽  
Paracrine Signalling ◽  
Cytokines And Chemokines

Abstract The pituitary gland is the master regulator of the endocrine system, housing six major hormone producing cell types. This gland is derived from Rathke’s Pouch, an invagination of the oral ectoderm. Hormone-producing pituitary cell lineages are derived from a population of embryonic cells expressing SOX2. ZFP36L1/Butyrate Response Factor 1 (BRF1) is an RNA binding protein that binds and targets mRNAs of various cytokines and chemokines for degradation prior to translation, attenuating secretion of inflammatory factors (Herranz et al. 2015). Here, we show that BRF1 is a novel marker expressed in SOX2+ cells in human and mouse pituitaries, suggesting that these cells may have a secretory profile. To investigate this possibility, we have combined molecular and genetic studies in vivo. We have used a novel mouse model, R26lsl-mBRF1 that allows the expression of a mutant, constitutively active BRF1 protein upon Cre-mediated recombination, alongside our lab’s models (Hesx1Cre/+ and Sox2CreERT2/+), to express mutant BRF1 in HESX1+ and SOX2+ cells during development and postnatally. This approach results in pituitary hypoplasia and severe hypopituitarism due to a failure of cell-lineage specified cells to differentiate into hormone-producing cells. Hormone production in these mutant cells, however, can be rescued in vitro through co-culture with WT pituitaries and in vivo in chimeric pituitaries, highlighting a cell non-autonomous mechanism underlying the phenotype. Single cell RNA sequencing of WT and Sox2CreERT2/+;R26lsl-mBRF1 murine embryonic pituitaries, as well as use publicly available human pituitary single cell datasets, have allowed us to identify specific cytokines and chemokines secreted by SOX2+ cells, as well as downstream intracellular signalling pathways in differentiating cells (Zhang et al. 2020), which may be responsible for controlling terminal differentiation of hormone-producing cells within the developing pituitary. Together with our recently published data, these results support the notion that SOX2+ pituitary stem cells play a critical paracrine role in controlling progenitor cell proliferation and terminal differentiation (Russell et al. 2021). References: Herranz, Nicolás et al. 2015. “MTOR Regulates MAPKAPK2 Translation to Control the Senescence-Associated Secretory Phenotype.” Nature Cell Biology 17(9): 1205–17. http://www.nature.com/doifinder/10.1038/ncb3225. Russell, John P et al. 2021. “Pituitary Stem Cells Produce Paracrine WNT Signals to Control the Expansion of Their Descendant Progenitor Cells.” eLife. Zhang, Shu et al. 2020. “Single-Cell Transcriptomics Identifies Divergent Developmental Lineage Trajectories during Human Pituitary Development.” Nature Communications.

scholarly journals Allele-resolved single-cell multi-omics uncovers the dynamics and transcriptional kinetics of X-chromosome upregulation

2021 ◽  
Author(s):  
Antonio Lentini ◽  
Huaitao Cheng ◽  
Joyce Carol Noble ◽  
Natali Papanicolaou ◽  
Christos Coucoravas ◽  
...  
Keyword(s):  
Single Cell ◽  
X Chromosome ◽  
Dosage Compensation ◽  
Chromatin Accessibility ◽  
Embryonic Cells ◽  
Mouse Development ◽  
Burst Frequency ◽  
Allelic Regulation

X-chromosome inactivation (XCI) and upregulation (XCU) are the major opposing chromosome-wide modes of gene regulation that collectively achieve dosage compensation in mammals, but the regulatory link between the two remains elusive. Here, we use allele-resolved single-cell RNA-seq combined with chromatin accessibility profiling to finely dissect the separate effects of XCI and XCU on RNA levels during mouse development. We uncover that balanced X dosage is flexibly attained through expression tuning by XCU in a sex- and lineage-specific manner along varying degrees of XCI and across developmental and cellular states. Male blastomeres achieve XCU upon zygotic genome activation while females experience two distinct waves of XCU, upon imprinted- and random XCI, and ablation of Xist impedes female XCU. Contrary to widely established models of mammalian dosage compensation, naïve female embryonic cells carrying two active X chromosomes do not exhibit upregulation but express both alleles at basal level, yet collectively exceeding the RNA output of a single hyperactive allele. We show, in vivo and in vitro, that XCU is kinetically driven by X-specific modulation of transcriptional burst frequency, coinciding with increased compartmentalization of the hyperactive allele. Altogether, our data provide unprecedented insights into the dynamics of mammalian XCU, prompting a revised model of the chain in events of allelic regulation by XCU and XCI in unitedly achieving stable cellular levels of X-chromosome transcripts.

scholarly journals Defining totipotency using criteria of increasing stringency

2020 ◽  
Author(s):  
Eszter Posfai ◽  
John Paul Schell ◽  
Adrian Janiszewski ◽  
Isidora Rovic ◽  
Alexander Murray ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Differentiated Cells ◽  
Totipotent Cell

AbstractTotipotency is the ability of a single cell to give rise to all the differentiated cells that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies upon a variety of assays of variable stringency. Here we describe criteria to define totipotency. We illustrate how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in the mouse, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbor increased totipotent potential relative to conventional embryonic stem cells under in vivo conditions.

Single-Cell Transcriptome of Early Hematopoiesis Guides Arterial Endothelium-Promoted Functional T Cell Generation from Human Pluripotent Stem Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 38-38
Author(s):  
Jun Shen ◽  
Yingxi Xu ◽  
Shuo Zhang ◽  
Shuzhen Lyu ◽  
Zack Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Hematopoietic Differentiation ◽  
Cell Potential ◽  
Arterial Endothelium

Human pluripotent stem cells (hPSCs) provide a powerful platform for generating functional hematopoietic cells for blood disease modeling and therapeutic testing. However, the quantity and quality of hPSC-derived blood cells remain to be improved. Here, by performing extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during hematopoietic differentiation of hPSCs, we construct the first hematopoietic landscape of hPSCs at the single-cell level and identify strategies to promote hematopoietic progenitor (HP) generation from hPSCs with functional T cell potential. By focusing specifically on cell populations and molecular events involved in endothelial-to-hematopoietic transition (EHT), we compared the difference of early hematopoiesis between hPSCs and human embryos (Yang Zeng et al. Cell Research. 2019) and found aerobic metabolism was dysregulated during in-vitro-directed differentiation. The decreased oxygen metabolism program was further deciphered as a key molecular event occurred during the EHT. Providing hypoxia at the stage of EHT enhanced hematopoietic differentiation of hPSCs via specifying arterial programs, including arterial hemogenic endothelium (AHE) and arterial endothelium cells (AE). To further determine the effect of AE on hematopoietic development, we isolated AE, venous endothelium and mesenchymal cells identified in our single-cell transcriptomic analyses and cocultured them with AHE respectively for HP generation. AE were finally validated as a critical regulator of definitive HP specification with more T cell potential. T cells generated from AE-primed HPs (AE-T) were highly functional and exhibited polyfunctional production of interferon (IFN)-γ, tumor necrosis factor alpha (TNF-α), and IL-2 in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin. To further evaluate the function of AE-T, we engineered T cells with CD19-CAR. The in vitro cytotoxicity of CAR-engineered AE-T was performed both in CD19+ cell lines (Nalm-6 and Raji) and human primary B-ALL samples. The efficacy of CAR-engineered AE-T in vivo was evaluated in a mouse xenograft model inoculated intravenously with luciferase-expressing Nalm-6 cells. Similar to CD19 CAR-transduced peripheral blood T cells, the AE-T potently inhibited tumor growth both in vitro and in vivo. Collectively, our study provides benchmark datasets to understand the origins of human hematopoiesis and presents an advance for guiding the generation of functional T cells in vitro for clinical applications. Disclosures No relevant conflicts of interest to declare.

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